Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats

ABSTRACT

The present invention provides systems, apparatuses, and methods to detect the presence of fetal cells when mixed with a population of maternal cells in a sample and to test fetal abnormalities, i.e. aneuploidy. In addition, the present invention provides methods to determine when there are insufficient fetal cells for a determination and report a non-informative case. The present invention involves quantifying regions of genomic DNA from a mixed sample. More particularly the invention involves quantifying DNA polymorphisms from the mixed sample.

CROSS-REFERENCE

This application is a Continuation of U.S. application Ser. No.16/819,992, filed on Mar. 16, 2020; which Continuation of U.S.application Ser. No. 13/830,871, filed on Mar. 14, 2013; which is aContinuation of U.S. application Ser. No. 13/738,268 filed Jan. 10,2013; which is a Continuation of U.S. application Ser. No. 13/433,232filed on Mar. 28, 2012; which is a Continuation of U.S. application Ser.No. 12/725,240 filed on Mar. 16, 2010; which is a Continuation of U.S.application Ser. No. 11/763,426 filed on Jun. 14, 2007; which claims thebenefit of U.S. Provisional Application Ser. No. 60/820,778 filed onJul. 28, 2006 and U.S. Provisional Application No. 60/804,815, filedJun. 14, 2006, which application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Analysis of specific cells can give insight into a variety of diseases.These analyses can provide non-invasive tests for detection, diagnosisand prognosis of diseases, thereby eliminating the risk of invasivediagnosis. For instance, social developments have resulted in anincreased number of prenatal tests. However, the available methodstoday, amniocentesis and chorionic villus sampling (CVS) are potentiallyharmful to the mother and to the fetus. The rate of miscarriage forpregnant women undergoing amniocentesis is increased by 0.5-1%, and thatfigure is slightly higher for CVS. Because of the inherent risks posedby amniocentesis and CVS, these procedures are offered primarily toolder women, i.e., those over 35 years of age, who have a statisticallygreater probability of bearing children with congenital defects. As aresult, a pregnant woman at the age of 35 has to balance an average riskof 0.5-1% to induce an abortion by amniocentesis against an age relatedprobability for trisomy 21 of less than 0.3%.

To eliminate the risks associated with invasive prenatal screeningprocedures, non-invasive tests for detection, diagnosis and prognosis ofdiseases, have been utilized. For example, maternal serumalpha-fetoprotein, and levels of unconjugated estriol and humanchorionic gonadotropin are used to identify a proportion of fetuses withDown's syndrome, however, these tests are not one hundred percentaccurate. Similarly, ultrasonography is used to determine congenitaldefects involving neural tube defects and limb abnormalities, but isuseful only after fifteen weeks' gestation.

The methods of the present invention allow for the detection of fetalcells and fetal abnormalities when fetal cells are present in a mixedpopulation of cells, even when maternal cells dominate the mixture.

SUMMARY OF THE INVENTION

The presence of fetal cells in maternal circulation offers theopportunity to develop a prenatal diagnostic that obviates the riskassociated with today's invasive diagnostics procedures. However, fetalcells are rare as compared to the presence of maternal cells in theblood. Therefore, any proposed analysis of fetal cells to diagnose fetalabnormalities requires enrichment of fetal cells. Enriching fetal cellsfrom maternal peripheral blood is challenging, time intensive and anyanalysis derived therefrom is prone to error. The present inventionaddresses these challenges.

The present invention relates to methods for determining the presence offetal cells and fetal abnormalities when fetal cells are present in amixed sample (e.g. maternal blood sample). In some embodiments,determining the presence of fetal cells or of a fetal abnormalityincludes comparing the level of genomic DNA from a mixed sample to thelevel of genomic DNA in a control sample. The control or referencesample can be a mixed sample that has been sufficiently diluted to befree of fetal cells. The mixed sample can contain at least one fetalcell and one non-fetal cell. In other embodiments, the sample comprisesup to 50% fetal cells.

In some embodiments, determining the presence of fetal cells and/orabnormalities involves quantifying one or more regions of genomic DNAregions from the mixed sample and determining from the quantificationthe presence of a fetal abnormality. Preferably, such regions arepolymorphic e.g. short tandem repeat (STR) regions.

Examples of fetal abnormalities that can be determined by quantifyingregions on one or more chromosomes include trisomy 13, trisomy 18,trisomy 21 (Down Syndrome), Klinefelter Syndrome (XXY) and otherirregular number of sex or autosomal chromosomes. Other examples ofabnormal fetal genotypes that can be determined by quantifying regionson one or more chromosomes include, but are not limited to, aneuploidysuch as, monosomy of one or more chromosomes (X chromosome monosomy,also known as Turner's syndrome), trisomy of one or more chromosomes(such as 13, 18, 21, and X), tetrasomy and pentasomy of one or morechromosomes (which in humans is most commonly observed in the sexchromosomes, e.g. XXXX, XXYY, XXXY, XYYY, XXXXX, XXXXY, XXXYY, XYYYY andXXYYY), triploidy (three of every chromosome, e.g. 69 chromosomes inhumans), tetraploidy (four of every chromosome, e.g. 92 chromosomes inhumans) and multiploidy. In some embodiments, an abnormal fetal genotypeis a segmental aneuploidy. Examples of segmental aneuploidy include, butare not limited to, 1p36 duplication, dup(17)(p11.2p11.2) syndrome, Downsyndrome, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome,and cat-eye syndrome. In some cases, an abnormal fetal genotype is dueto one or more deletions of sex or autosomal chromosomes, which mayresult in a condition such as Cri-du-chat syndrome, Wolf-Hirschhorn,Williams-Beuren syndrome, Charcot-Marie-Tooth disease, Hereditaryneuropathy with liability to pressure palsies, Smith-Magenis syndrome,Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, Steroid sulfatase deficiency, Kallmann syndrome,Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, Testis-determiningfactor on Y, Azospermia (factor a), Azospermia (factor b), Azospermia(factor c), or 1p36 deletion. In some embodiments, a decrease inchromosomal number results in an XO syndrome.

Furthermore, the methods herein can distinguish maternal trisomy frompaternal trisomy, and total aneuploidy from segmental aneuploidy.Segmental aneuploidies can be caused by an intra-chromosomal event suchas a deletion, duplication or translocation event. Additionally, themethods herein can be used to identify monoploidy, triploidy,tetraploidy, pentaploidy and other higher multiples of the normalhaploid state. In some embodiments, the maternal or paternal origin ofthe fetal abnormality can be determined.

The genomic DNA region(s) can be quantified by amplifying the regionsusing, for example, PCR, or preferably quantitative PCR. Alternatively,quantification of the regions can be achieved using capillary gelelectrophoresis (CGE). In some embodiments, total genomic DNA ispre-amplified prior to the quantitative amplification step to increasethe overall abundance of DNA. Such pre-amplification step can involvethe use of multiple displacement amplification.

In some embodiments the genomic DNA regions quantified can be in onechromosome or in 2 or more chromosomes. The polymorphic regions can bequantified on either or both sex chromosomes X and Y, and on autosomalchromosomes including chromosomes 13, 18 and 21.

Prior to analysis a mixed sample suspected of having fetal cells (e.g. amaternal blood sample) can be enriched for fetal cells. Fetal cellenrichment can be accomplished using any method known in the artincluding size-based separation, affinity (e.g. magnetic) separation,FACS, laser microdisection, and magnetic bead separation. A mixed samplecontaining as few as 10 fetal cells can be enriched. In someembodiments, the fetal cells in the enriched sample constitute less than50% of the total number of cells.

In some embodiments, the size-based separation method includes applyinga mixed sample into a system that separates a first component of themixed sample (e.g. fetal cells), which comprises cells that are largerthan a critical size, in a first direction, and a second component ofthe mixed sample (e.g. enucleated maternal red blood cells), whichcomprises cells that are smaller than a critical size, towards a secondexit port. The separation system can be a device that includes one ormore arrays of obstacles that form a network of gaps.

In some embodiments, enrichment that is achieved by size-basedseparation is followed by one or more additional enrichment proceduresincluding magnetic separation, fluorescence activated cell sorting(FACS), laser microdisection, and magnetic bead separation. In someembodiments, a sample enriched by size-based separation is subjected toaffinity/magnetic separation and is further enriched for rare cellsusing fluorescence activated cell sorting (FACS) or selective lysis of asubset of the cells (e.g. fetal cells).

In some embodiments there are provided kits for detecting the fetalabnormalities wherein the kits include separation devices and thereagents needed to perform the genetic analysis. For example, the kitmay include arrays for size based enrichment, a device for magneticenrichment and reagents for performing PCR.

The methods can further comprise inputting the data from thequantification step into data model(s) for the association of DNAquantity with maternal and non-maternal alleles. The invention providesfor a computer program product, which includes a computer executablelogic recorded on a computer readable medium that can be used fordiagnosing a fetal abnormality. The computer program is designed toreceive data from one of more quantified DNA genomic regions from amixed sample containing at least one fetal cell, determine the presenceor absence of a fetal abnormality from the data, and generate an outputthat comprises the evaluation of the fetal abnormality. Methods forusing the computer program product are also disclosed.

SUMMARY OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a flow chart of one embodiment of the presentinvention.

FIGS. 2A-2D illustrate one embodiment of a size-based separation module.

FIGS. 3A-3C illustrate one embodiment of an affinity separation module.

FIG. 4 illustrates one embodiment of a magnetic separation module.

FIG. 5 illustrates typical locus patterns arising from a normal(diploid) fetus and mother.

FIG. 6 illustrates typical locus patterns arising from trisomic fetalcells.

FIGS. 7A-7D illustrate various embodiments of a size-based separationmodule.

FIGS. 8A-8B illustrate cell smears of the product and waste fractions.

FIGS. 9A-9F illustrate isolated fetal cells confirmed by the reliablepresence of male Y chromosome.

FIG. 10 illustrates trisomy 21 pathology in an isolated fetal nucleatedred blood cell.

FIG. 11 depicts a flow chart depicting the major steps involved indetecting paternal alleles in a fetal enriched sample usingfluorescently labeled primers.

FIG. 12 illustrates a table with STR loci that can be used for fetaldetection.

FIG. 13 illustrates a table with exemplary external primers for STRloci.

FIG. 14 illustrates a table with exemplary internal primers for STRloci.

FIG. 15 illustrates the resolution for the ABI 310 bioanalyzer.

FIG. 16 illustrates the detection limit on fixed cord blood.

FIG. 17 illustrates the detection of 10 fetal cells at 10% puritywithout nested PCR.

FIG. 18 illustrates the generation of STR markers on fixed cellsrecovered from a slide.

FIG. 19 illustrates detection of fetal alleles at less than 10% purityafter nested PCR amplification of STRs.

FIG. 20 illustrates the detection of single copies of a fetal cellgenome by qPCR.

FIG. 21 illustrates detection of single fetal cells in binned samples bySNP analysis.

FIG. 22 illustrates a method of trisomy testing. The trisomy 21 screenis based on scoring of target cells obtained from maternal blood. Bloodis processed using a cell separation module for hemoglobin enrichment(CSM-HE). Enriched cells are transferred to slides that are firststained and subsequently probed by FISH. Images are acquired, such asfrom bright field or fluorescent microscopy, and scored. The proportionof trisomic cells of certain classes serves as a classifier for risk offetal trisomy 21. Fetal genome identification can performed using assayssuch as: (1) STR markers; (2) qPCR using primers and probes directed toloci, such as the multi-repeat DYZ locus on the Y-chromosome; (3) SNPdetection; and (4) CGH (comparative genome hybridization) arraydetection.

FIG. 23 illustrates assays that can produce information on the presenceof aneuploidy and other genetic disorders in target cells. Informationon anueploidy and other genetic disorders in target cells may beacquired using technologies such as: (1) a CGH array established forchromosome counting, which can be used for aneuploidy determinationand/or detection of intra-chromosomal deletions; (2) SNP/taqman assays,which can be used for detection of single nucleotide polymorphisms; and(3) ultra-deep sequencing, which can be used to produce partial orcomplete genome sequences for analysis.

FIG. 24 illustrates methods of fetal diagnostic assays. Fetal cells areisolated by CSM-HE enrichment of target cells from blood. Thedesignation of the fetal cells may be confirmed using techniquescomprising FISH staining (using slides or membranes and optionally anautomated detector), FACS, and/or binning. Binning may comprisedistribution of enriched cells across wells in a plate (such as a 96 or384 well plate), microencapsulation of cells in droplets that areseparated in an emulsion, or by introduction of cells into microarraysof nanofluidic bins. Fetal cells are then identified using methods thatmay comprise the use of biomarkers (such as fetal (gamma) hemoglobin),allele-specific SNP panels that could detect fetal genome DNA, detectionof differentially expressed maternal and fetal transcripts (such asAffymetrix chips), or primers and probes directed to fetal specific loci(such as the multi-repeat DYZ locus on the Y-chromosome). Binning sitesthat contain fetal cells are then be analyzed for aneuploidy and/orother genetic defects using a technique such as CGH array detection,ultra deep sequencing (such as Solexa, 454 or mass spectrometry), STRanalysis, or SNP detection.

FIG. 25 illustrates methods of fetal diagnostic assays, furthercomprising the step of whole genome amplification prior to analysis ofaneuploidy and/or other genetic defects.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides systems, apparatuses, and methods todetect the presence and condition (e.g. aneuploidy) of fetal cells in amixed cell population, e.g. a sample wherein fetal cells consist of<50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0.5% of allcells in a mixed sample.

FIG. 1 illustrates an overview of one embodiment of the presentinvention.

In step 100, a sample containing (or suspected of containing) 1 or morefetal cells is obtained. Samples can be obtained from an animalsuspected of being pregnant, pregnant, or that has been pregnant todetect the presence of a fetus or fetal abnormality. Such animal can bea human or a domesticated animal such as a cow, chicken, pig, horse,rabbit, dog, cat, or goat. Samples derived from an animal or human caninclude, e.g., whole blood, sweat, tears, ear flow, sputum, lymph, bonemarrow suspension, lymph, urine, saliva, semen, vaginal flow,cerebrospinal fluid, brain fluid, ascites, milk, secretions of therespiratory, intestinal or genitourinary tracts fluid.

To obtain a blood sample, any technique known in the art may be used,e.g. a syringe or other vacuum suction device. A blood sample can beoptionally pre-treated or processed prior to enrichment. Examples ofpre-treatment steps include the addition of a reagent such as astabilizer, a preservative, a fixant, a lysing reagent, a diluent, ananti-apoptotic reagent, an anti-coagulation reagent, an anti-thromboticreagent, magnetic property regulating reagent, a buffering reagent, anosmolality regulating reagent, a pH regulating reagent, and/or across-linking reagent.

When a blood sample is obtained, a preservative such an anti-coagulationagent and/or a stabilizer can be added to the sample prior toenrichment. This allows for extended time for analysis/detection. Thus,a sample, such as a blood sample, can be enriched and/or analyzed underany of the methods and systems herein within 1 week, 6 days, 5 days, 4days, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr fromthe time the sample is obtained.

In some embodiments, a blood sample can be combined with an agent thatselectively lyses one or more cells or components in a blood sample. Forexample, fetal cells can be selectively lysed releasing their nucleiwhen a blood sample including fetal cells is combined with deionizedwater. Such selective lysis allows for the subsequent enrichment offetal nuclei using, e.g., size or affinity based separation. In anotherexample, platelets and/or enucleated red blood cells are selectivelylysed to generate a sample enriched in nucleated cells, such as fetalnucleated red blood cells (fnRBC) and maternal nucleated blood cells(mnBC). The fnRBC's can subsequently be separated from the mnBC's using,e.g., affinity to antigen-i or magnetism differences in fetal and adulthemoglobin.

When obtaining a sample from an animal (e.g., blood sample), the amountcan vary depending upon animal size, its gestation period, and/or thecondition being screened. In some embodiments, up to 50, 40, 30, 20, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 mL of a sample is obtained. In someembodiments, 1-50, 2-40, 3-30, or 4-20 mL of sample is obtained. In someembodiments, more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100 mL of a sample is obtained.

To detect fetal abnormality, a blood sample can be obtained from apregnant animal or human within 36, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6or 4 weeks of gestation.

In step 101, a reference sample is obtained. The reference sampleconsists of substantially all or all maternal cells. In someembodiments, a reference sample is a maternal blood sample enriched forwhite blood cells (WBC's) such that it consists of substantially all orall maternal WBC's. In some embodiments, a reference sample is a dilutedmixed sample wherein the dilution results in a sample free of fetalcells. For example, a maternal blood sample of 10-50 ML can be dilutedby at least 2, 5, 10, 20, 50, or 100 fold to reduce the likelihood thatit will include fetal cells.

In step 102, when the sample to be tested or analyzed is a mixed sample(e.g. maternal blood sample), it is enriched for rare cells or rare DNA(e.g. fetal cells, fetal DNA or fetal nuclei) using one or more methodsknown in the art or disclosed herein. Such enrichment increases theratio of fetal cells to non-fetal cells; the concentration of fetal DNAto non-fetal DNA; or the concentration of fetal cells in volume pertotal volume of the mixed sample.

In some embodiments, enrichment occurs by selective lysis as describedabove. For example, enucleated cells may be selectively lysed prior tosubsequent enrichment steps or fetal nucleated cells may be selectivelylysed prior to separation of the fetal nuclei from other cells andcomponents in the sample.

In some embodiments, enrichment of fetal cells or fetal nuclei occursusing one or more size-based separation modules. Size-based separationmodules include filtration modules, sieves, matrixes, etc., includingthose disclosed in International Publication Nos. WO 2004/113877, WO2004/0144651, and US Application Publication No. 2004/011956.

In some embodiments, a size-based separation module includes one or morearrays of obstacles that form a network of gaps. The obstacles areconfigured to direct particles (e.g. cells or nuclei) as they flowthrough the array/network of gaps into different directions or outletsbased on the particle's hydrodynamic size. For example, as a bloodsample flows through an array of obstacles, nucleated cells or cellshaving a hydrodynamic size larger than a critical size, e.g., 8 microns,are directed to a first outlet located on the opposite side of the arrayof obstacles from the fluid flow inlet, while the enucleated cells orcells having a hydrodynamic size smaller than a critical size, e.g., 8microns, are directed to a second outlet also located on the oppositeside of the array of obstacles from the fluid flow inlet.

An array can be configured to separate cells smaller than a criticalsize from those larger than the critical size by adjusting the size ofthe gaps, obstacles, and offset in the period between each successiverow of obstacles. For example, in some embodiments, obstacles and/orgaps between obstacles can be up to 10, 20, 50, 70, 100, 120, 150, 170,or 200 microns in length or about 2, 4, 6, 8 or 10 microns in length. Insome embodiments, an array for size-based separation includes more than100, 500, 1,000, 5,000, 10,000, 50,000 or 100,000 obstacles that arearranged into more than 10, 20, 50, 100, 200, 500, or 1000 rows.Preferably, obstacles in a first row of obstacles are offset from aprevious (upstream) row of obstacles by up to 50% of the period of theprevious row of obstacles. In some embodiments, obstacles in a first rowof obstacles are offset from a previous row of obstacles by up to 45,40, 35, 30, 25, 20, 15 or 10% the period of the previous row ofobstacles. Furthermore, the distance between a first row of obstaclesand a second row of obstacles can be up to 10, 20, 50, 70, 100, 120,150, 170 or 200 microns. A particular offset can be continuous(repeating for multiple rows) or non-continuous. In some embodiments, aseparation module includes multiple discrete arrays of obstacles fluidlycoupled such that they are in series with one another. Each array ofobstacles has a continuous offset. But each subsequent (downstream)array of obstacles has an offset that is different from the previous(upstream) offset. Preferably, each subsequent array of obstacles has asmaller offset that the previous array of obstacles. This allows for arefinement in the separation process as cells migrate through the arrayof obstacles. Thus, a plurality of arrays can be fluidly coupled inseries or in parallel, (e.g., more than 2, 4, 6, 8, 10, 20, 30, 40, 50).Fluidly coupling separation modules (e.g., arrays) in parallel allowsfor high-throughput analysis of the sample, such that at least 1, 2, 5,10, 20, 50, 100, 200, or 500 mL per hour flows through the enrichmentmodules or at least 1, 5, 10, or 50 million cells per hour are sorted orflow through the device.

FIGS. 2A-2D illustrate one example of a size-based separation module.Obstacles (which may be of any shape) are coupled to a flat substrate toform an array of gaps. A transparent cover or lid may be used to coverthe array. The obstacles form a two-dimensional array with eachsuccessive row shifted horizontally with respect to the previous row ofobstacles, where the array of obstacles directs components having ahydrodynamic size smaller than a critical size in a first direction andcomponents having a hydrodynamic size larger that a critical size in asecond direction. See FIGS. 2B-2D. The flow of sample into the array ofobstacles can be aligned at a small angle (flow angle) with respect to aline-of-sight of the array (lateral flow angle). Optionally, the arrayis coupled to an infusion pump to perfuse the sample through theobstacles. The flow conditions of the size-based separation moduledescribed herein are such that cells are sorted by the array withminimal damage. This allows for downstream analysis of intact cells andintact nuclei to be more efficient and reliable.

In one embodiment, a size-based separation module comprises an array ofobstacles configured to direct rare cells larger than a critical size tomigrate along a line-of-sight within the array towards a first outlet orbypass channel leading to a first outlet, while directing cells andanalytes smaller than a critical size through the array of obstacles ina different direction towards a second outlet.

A variety of enrichment protocols may be utilized although gentlehandling of the cells is preferred to reduce any mechanical damage tothe cells or their DNA. This gentle handling also preserves the smallnumber of fetal cells in the sample. Integrity of the nucleic acid beingevaluated is an important feature in some embodiments to permit thedistinction between the genomic material from the fetal cells and othercells in the sample. In particular, the enrichment and separation of thefetal cells using the arrays of obstacles produces gentle treatmentwhich minimizes cellular damage and maximizes nucleic acid integritypermitting exceptional levels of separation and the ability tosubsequently utilize various formats to very accurately analyze thegenome of the cells which are present in the sample in extremely lownumbers.

In some embodiments, enrichment of fetal cells occurs using one or morecapture modules that selectively inhibit the mobility of one or morecells of interest. Preferably a capture module is fluidly coupleddownstream to a size-based separation module. Capture modules caninclude a substrate having multiple obstacles that restrict the movementof cells or analytes greater than a critical size. Examples of capturemodules that inhibit the migration of cells based on size are disclosedin U.S. Pat. Nos. 5,837,115 and 6,692,952.

In some embodiments, a capture module includes a two dimensional arrayof obstacles that selectively filters or captures cells or analyteshaving a hydrodynamic size greater than a particular gap size, e.g.,critical sized. Arrays of obstacles adapted for separation by capturecan include obstacles having one or more shapes and can be arranged in auniform or non-uniform order. In some embodiments, a two-dimensionalarray of obstacles is staggered such that each subsequent row ofobstacles is offset from the previous row of obstacles to increase thenumber of interactions between the analytes being sorted (separated) andthe obstacles.

Another example of a capture module is an affinity-based separationmodule. An affinity-based separation module capture analytes or cells ofinterest based on their affinity to a structure or particle as oppose totheir size. One example of an affinity-based separation module is anarray of obstacles that are adapted for complete sample flow through,but for the fact that the obstacles are covered with binding moietiesthat selectively bind one or more analytes (e.g., cell population) ofinterest (e.g., red blood cells, fetal cells, or nucleated cells) oranalytes not-of-interest (e.g., white blood cells). Binding moieties caninclude e.g., proteins (e.g., ligands/receptors), nucleic acids havingcomplementary counterparts in retained analytes, antibodies, etc. Insome embodiments, an affinity-based separation module comprises atwo-dimensional array of obstacles covered with one or more antibodiesselected from the group consisting of: anti-CD71, anti-CD235a,anti-CD36, anti-carbohydrates, anti-selectin, anti-CD45, anti-GPA, andanti-antigen-i.

FIG. 3A illustrates a path of a first analyte through an array of postswherein an analyte that does not specifically bind to a post continuesto migrate through the array, while an analyte that does bind a post iscaptured by the array. FIG. 3B is a picture of antibody coated posts.FIG. 3C illustrates one method of coupling of antibodies to a substrate(e.g., obstacles, side walls, etc.) as contemplated by the presentinvention. Examples of such affinity-based separation modules aredescribed in International Publication No. WO 2004/029221 and U.S.application Ser. No. 10/529,453, both of which are incorporated byreference.

In some embodiments, a capture module utilizes a magnetic field toseparate and/or enrich one or more analytes (cells) that has a magneticproperty or magnetic potential. For example, red blood cells which areslightly diamagnetic (repelled by magnetic field) in physiologicalconditions can be made paramagnetic (attributed by magnetic field) bydeoxygenation of the hemoglobin into methemoglobin. This magneticproperty can be achieved through physical or chemical treatment of thered blood cells. Thus, a sample containing one or more red blood cellsand one or more non-red blood cells can be enriched for the red bloodcells by first inducing a magnetic property and then separating theabove red blood cells from other analytes using a magnetic field(uniform or non-uniform). For example, a maternal blood sample can flowfirst through a size-based separation module to remove enucleated cellsand cellular components (e.g., analytes having a hydrodynamic size lessthan 6 μms) based on size. Subsequently, the enriched nucleated cells(e.g., analytes having a hydrodynamic size greater than 6 μms) whiteblood cells and nucleated red blood cells are treated with a reagent,such as CO₂, N₂ or NaNO₂, that changes the magnetic property of the redblood cells' hemoglobin. The treated sample then flows through amagnetic field (e.g., a column coupled to an external magnet), such thatthe paramagnetic analytes (e.g., red blood cells) will be captured bythe magnetic field while the white blood cells and any other non-redblood cells will flow through the device to result in a sample enrichedin nucleated red blood cells (including fnRBC's). Additional examples ofmagnetic separation modules are described in U.S. application Ser. No.11/323,971, filed Dec. 29, 2005 entitled “Devices and Methods forMagnetic Enrichment of Cells and Other Particles” and U.S. applicationSer. No. 11/227,904, filed Sep. 15, 2005, entitled “Devices and Methodsfor Enrichment and Alteration of Cells and Other Particles”.

Subsequent enrichment steps can be used to separate the rare cells (e.g.fnRBC's) from the non-rare maternal nucleated red blood cells(non-RBC's). In some embodiments, a sample enriched by size-basedseparation followed by affinity/magnetic separation is further enrichedfor rare cells using fluorescence activated cell sorting (FACS) orselective lysis of a subset of the cells (e.g. fetal cells). In someembodiments, fetal cells are selectively bound to an anti-antigen ibinding moiety (e.g. an antibody) to separate them from the mnRBC's. Insome embodiments, the antibody binds to a fetal cell ligand. In somerelated embodiments the fetal cells are stimulated so as to induceexpression of ligands which are targeted by an antibody. In someembodiments the fetal cells are lysed and the nuclei of the fetal cellsare separated from other cellular components by binding them with anantobody. In some embodiments, fetal cells are selectively bound toreceptors which target fetal cell ligands. In some embodiments, fetalcells are selectively bound to a lectin. In some embodiments, fetalcells or fetal DNA is distinguished from non-fetal cells or non-fetalDNA by forcing the rare cells (fetal cells) to become apoptotic, thuscondensing their nuclei and optionally ejecting their nuclei. Rare cellssuch as fetal cells can be forced into apoptosis using various meansincluding subjecting the cells to hyperbaric pressure (e.g. 4% CO₂). Thecondensed nuclei can be detected and/or isolated for further analysisusing any technique known in the art including DNA gel electrophoresis,in situ labeling of DNA nicks (terminal deoxynucleotidyl transferase(TdT))-mediated dUTP in situ nick labeling (also known as TUNEL)(Gavrieli, Y., et al. J. Cell Biol 119:493-501 (1992)) and ligation ofDNA strand breaks having one or two-base 3′ overhangs (Taqpolymerase-based in situ ligation). (Didenko V., et al. J. Cell Biol.135:1369-76 (1996)).

In some embodiments, when the analyte desired to be separated (e.g., redblood cells or white blood cells) is not ferromagnetic or does not havea magnetic property, a magnetic particle (e.g., a bead) or compound(e.g., Fe³⁺) can be coupled to the analyte to give it a magneticproperty. In some embodiments, a bead coupled to an antibody thatselectively binds to an analyte of interest can be decorated with anantibody elected from the group of anti CD71 or CD75. In someembodiments a magnetic compound, such as Fe³⁺, can be couple to anantibody such as those described above. The magnetic particles ormagnetic antibodies herein may be coupled to any one or more of thedevices herein prior to contact with a sample or may be mixed with thesample prior to delivery of the sample to the device(s).

Magnetic field used to separate analytes/cells in any of the embodimentsherein can uniform or non-uniform as well as external or internal to thedevice(s) herein. An external magnetic field is one whose source isoutside a device herein (e.g., container, channel, obstacles). Aninternal magnetic field is one whose source is within a devicecontemplated herein. An example of an internal magnetic field is onewhere magnetic particles may be attached to obstacles present in thedevice (or manipulated to create obstacles) to increase surface area foranalytes to interact with to increase the likelihood of binding.Analytes captured by a magnetic field can be released by demagnetizingthe magnetic regions retaining the magnetic particles. For selectiverelease of analytes from regions, the demagnetization can be limited toselected obstacles or regions. For example, the magnetic field can bedesigned to be electromagnetic, enabling turn-on and turn-off off themagnetic fields for each individual region or obstacle at will.

FIG. 4 illustrates an embodiment of a device configured for capture andisolation of cells expressing the transferrin receptor from a complexmixture. Monoclonal antibodies to CD71 receptor are readily availableoff-the-shelf and can be covalently coupled to magnetic materialscomprising any conventional ferroparticles, such as, but not limited toferrous doped polystyrene and ferroparticles or ferro-colloids (e.g.,from Miltenyi or Dynal). The anti CD71 bound to magnetic particles isflowed into the device. The antibody coated particles are drawn to theobstacles (e.g., posts), floor, and walls and are retained by thestrength of the magnetic field interaction between the particles and themagnetic field. The particles between the obstacles and those looselyretained with the sphere of influence of the local magnetic fields awayfrom the obstacles are removed by a rinse.

One or more of the enrichment modules herein (e.g., size-basedseparation module(s) and capture module(s)) may be fluidly coupled inseries or in parallel with one another. For example a first outlet froma separation module can be fluidly coupled to a capture module. In someembodiments, the separation module and capture module are integratedsuch that a plurality of obstacles acts both to deflect certain analytesaccording to size and direct them in a path different than the directionof analyte(s) of interest, and also as a capture module to capture,retain, or bind certain analytes based on size, affinity, magnetism orother physical property.

In any of the embodiments herein, the enrichment steps performed have aspecificity and/or sensitivity ≥50, 60, 70, 80, 90, 95, 96, 97, 98, 99,99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 99.95% Theretention rate of the enrichment module(s) herein is such that ≥50, 60,70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% of the analytesor cells of interest (e.g., nucleated cells or nucleated red blood cellsor nucleated from red blood cells) are retained. Simultaneously, theenrichment modules are configured to remove ≥50, 60, 70, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 99.9% of all unwanted analytes (e.g.,red blood-platelet enriched cells) from a sample.

Any or all of the enrichment steps can occur with minimal dilution ofthe sample. For example, in some embodiments the analytes of interestare retained in an enriched solution that is less than 50, 40, 30, 20,10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or0.5 fold diluted from the original sample. In some embodiments, any orall of the enrichment steps increase the concentration of the analyte ofinterest (fetal cell), for example, by transferring them from the fluidsample to an enriched fluid sample (sometimes in a new fluid medium,such as a buffer). The new concentration of the analyte of interest maybe at least 2, 4, 6, 8, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000,10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000,5,000,000, 10,000,000, 20,000,000, 50,000,000, 100,000,000, 200,000,000,500,000,000, 1,000,000,000, 2,000,000,000, or 5,000,000,000 fold moreconcentrated than in the original sample. For example, a 10 timesconcentration increase of a first cell type out of a blood sample meansthat the ratio of first cell type/all cells in a sample is 10 timesgreater after the sample was applied to the apparatus herein. Suchconcentration can take a fluid sample (e.g., a blood sample) of greaterthan 10, 15, 20, 50, or 100 mL total volume comprising rare componentsof interest, and it can concentrate such rare component of interest intoa concentrated solution of less than 0.5, 1, 2, 3, 5, or 10 mL totalvolume.

The final concentration of fetal cells in relation to non-fetal cellsafter enrichment can be about 1/10,000-1/10, or 1/1,000-1/100. In someembodiments, the concentration of fetal cells to maternal cells may beup to 1/1,000, 1/100, or 1/10 or as low as 1/100, 1/1,000 or 1/10,000.

Thus, detection and analysis of the fetal cells can occur even if thenon-fetal (e.g. maternal) cells are >50%, 60%, 70%, 80%, 90%, 95%, or99% of all cells in a sample. In some embodiments, fetal cells are at aconcentration of less than 1:2, 1:4, 1:10, 1:50, 1:100, 1:1000,1:10,000, 1:100,000, 1,000,000, 1:10,000,000 or 1:100,000,000 of allcells in a mixed sample to be analyzed or at a concentration of lessthan 1×10⁻³, 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶, or 1×10⁻⁶ cells/μL of the mixedsample. Over all, the number of fetal cells in a mixed sample, (e.g.enriched sample) has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 100 total fetal cells.

Enriched target cells (e.g., fnRBC) may be “binned” prior to furtheranalysis of the enriched cells (FIGS. 24 and 25 ). Binning is anyprocess which results in the reduction of complexity and/or total cellnumber of the enriched cell output. Binning may be performed by anymethod known in the art or described herein. One method of binning is byserial dilution. Such dilution may be carried out using any appropriateplatform (e.g., PCR wells, microtiter plates) and appropriate buffers.Other methods include nanofluidic systems which can separate samplesinto droplets (e.g., BioTrove, Raindance, Fluidigm). Such nanofluidicsystems may result in the presence of a single cell present in ananodroplet.

Binning may be preceded by positive selection for target cellsincluding, but not limited to, affinity binding (e.g. using anti-CD71antibodies). Alternately, negative selection of non-target cells mayprecede binning. For example, output from a size-based separation modulemay be passed through a magnetic hemoglobin enrichment module (MHEM)which selectively removes WBCs from the enriched sample by attractingmagnetized hemoglobin-containing cells.

For example, the possible cellular content of output from enrichedmaternal blood which has been passed through a size-based separationmodule (with or without further enrichment by passing the enrichedsample through a MHEM) may consist of: 1) approximately 20 fnRBC; 2)1,500 mnRBC; 3) 4,000-40,000 WBC; 4) 15×10⁶ RBC. If this sample isseparated into 100 bins (PCR wells or other acceptable binningplatform), each bin would be expected to contain: 1) 80 negative binsand 20 bins positive for one fnRBC; 2) 150 mnRBC; 3) 400-4,000 WBC; 4)15×10⁴ RBC. If separated into 10,000 bins, each bin would be expected tocontain: 1) 9,980 negative bins and 20 bins positive for one fnRBC; 2)8,500 negative bins and 1,500 bins positive for one mnRBC; 3)<1-4 WBC;4) 15×10² RBC. One of skill in the art will recognize that the number ofbins may be increased or decreased depending on experimental designand/or the platform used for binning. Reduced complexity of the binnedcell populations may facilitate further genetic and/or cellular analysisof the target cells by reducing the number of non-target cells in anindividual bin.

Analysis may be performed on individual bins to confirm the presence oftarget cells (e.g. fnRBC) in the individual bin. Such analysis mayconsist of any method known in the art including, but not limited to,FISH, PCR, STR detection, SNP analysis, biomarker detection, andsequence analysis (FIGS. 24 and 25 ).

Fetal Biomarkers

In some embodiments fetal biomarkers may be used to detect and/orisolate fetal cells, after enrichment or after detection of fetalabnormality or lack thereof. For example, this may be performed bydistinguishing between fetal and maternal nRBCs based on relativeexpression of a gene (e.g., DYS1, DYZ, CD-71, ε- and ζ-globin) that isdifferentially expressed during fetal development. In preferredembodiments, biomarker genes are differentially expressed in the firstand/or second trimester. “Differentially expressed,” as applied tonucleotide sequences or polypeptide sequences in a cell or cell nuclei,refers to differences in over/under-expression of that sequence whencompared to the level of expression of the same sequence in anothersample, a control or a reference sample. In some embodiments, expressiondifferences can be temporal and/or cell-specific. For example, forcell-specific expression of biomarkers, differential expression of oneor more biomarkers in the cell(s) of interest can be higher or lowerrelative to background cell populations. Detection of such difference inexpression of the biomarker may indicate the presence of a rare cell(e.g., fnRBC) versus other cells in a mixed sample (e.g., backgroundcell populations). In other embodiments, a ratio of two or more suchbiomarkers that are differentially expressed can be measured and used todetect rare cells.

In one embodiment, fetal biomarkers comprise differentially expressedhemoglobins. Erythroblasts (nRBCs) are very abundant in the early fetalcirculation, virtually absent in normal adult blood and by having ashort finite lifespan, there is no risk of obtaining fnRBC which maypersist from a previous pregnancy. Furthermore, unlike trophoblastcells, fetal erythroblasts are not prone to mosaic characteristics.

Yolk sac erythroblasts synthesize ε-, ζ-, γ- and α-globins, thesecombine to form the embryonic hemoglobins. Between six and eight weeks,the primary site of erythropoiesis shifts from the yolk sac to theliver, the three embryonic hemoglobins are replaced by fetal hemoglobin(HbF) as the predominant oxygen transport system, and ε- and ζ-globinproduction gives way to γ-, α- and β-globin production within definitiveerythrocytes (Peschle et al., 1985). HbF remains the principalhemoglobin until birth, when the second globin switch occurs andβ-globin production accelerates.

Hemoglobin (Hb) is a heterodimer composed of two identical a globinchains and two copies of a second globin. Due to differential geneexpression during fetal development, the composition of the second chainchanges from c globin during early embryonic development (1 to 4 weeksof gestation) to γ globin during fetal development (6 to 8 weeks ofgestation) to β globin in neonates and adults as illustrated in (Table1).

TABLE 1 Relative expression of ε, γ and β in maternal and fetal RBCs. εγ B 1^(st) trimester Fetal ++ ++ − Maternal − +/− ++ 2^(nd) trimesterFetal − ++ +/− Maternal − +/− ++

In the late-first trimester, the earliest time that fetal cells may besampled by CVS, fnRBCs contain, in addition to α globin, primarily ε andγ globin. In the early to mid second trimester, when amniocentesis istypically performed, fnRBCs contain primarily γ globin with some adult βglobin. Maternal cells contain almost exclusively α and β globin, withtraces of γ detectable in some samples. Therefore, by measuring therelative expression of the ε, γ and β genes in RBCs purified frommaternal blood samples, the presence of fetal cells in the sample can bedetermined. Furthermore, positive controls can be utilized to assessfailure of the FISH analysis itself.

In various embodiments, fetal cells are distinguished from maternalcells based on the differential expression of hemoglobins β, γ or ε.Expression levels or RNA levels can be determined in the cytoplasm or inthe nucleus of cells. Thus in some embodiments, the methods hereininvolve determining levels of messenger RNA (mRNA), ribosomal RNA(rRNA), or nuclear RNA (nRNA).

In some embodiments, identification of fnRBCs can be achieved bymeasuring the levels of at least two hemoglobins in the cytoplasm ornucleus of a cell. In various embodiments, identification and assay isfrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fetal nuclei. Furthermore,total nuclei arrayed on one or more slides can number from about 100,200, 300, 400, 500, 700, 800, 5000, 10,000, 100,000, 1,000,000,2,000,000 to about 3,000,000. In some embodiments, a ratio for γ/β orε/β is used to determine the presence of fetal cells, where a numberless than one indicates that a fnRBC(s) is not present. In someembodiments, the relative expression of γ/β or ε/β provides a fnRBCindex (“FNI”), as measured by γ or ε relative to (3. In someembodiments, a FNI for γ/β greater than 5, 10, 15, 20, 25, 30, 35, 40,45, 90, 180, 360, 720, 975, 1020, 1024, 1250 to about 1250, indicatethat a fnRBC(s) is present. In yet other embodiments, a FNI for γ/β ofless than about 1 indicates that a fnRBC(s) is not present. Preferably,the above FNI is determined from a sample obtained during a firsttrimester. However, similar ratios can be used during second trimesterand third trimester.

In some embodiments, the expression levels are determined by measuringnuclear RNA transcripts including, nascent or unprocessed transcripts.In another embodiment, expression levels are determined by measuringmRNA, including ribosomal RNA. There are many methods known in the artfor imaging (e.g., measuring) nucleic acids or RNA including, but notlimited to, using expression arrays from Affymetrix, Inc. or Illumina,Inc.

RT-PCR primers can be designed by targeting the globin variable regions,selecting the amplicon size, and adjusting the primers annealingtemperature to achieve equal PCR amplification efficiency. Thus TaqManprobes can be designed for each of the amplicons with well-separatedfluorescent dyes, Alexa Fluor®-355 for ε, Alexa Fluor®-488 for γ, andAlexa Fluor-555 for β. The specificity of these primers can be firstverified using ε, γ, and β cDNA as templates. The primer sets that givethe best specificity can be selected for further assay development. Asan alternative, the primers can be selected from two exons spanning anintron sequence to amplify only the mRNA to eliminate the genomic DNAcontamination.

The primers selected can be tested first in a duplex format to verifytheir specificity, limit of detection, and amplification efficiencyusing target cDNA templates. The best combinations of primers can befurther tested in a triplex format for its amplification efficiency,detection dynamic range, and limit of detection.

Various commercially available reagents are available for RT-PCR, suchas One-step RT-PCR reagents, including Qiagen One-Step RT-PCR Kit andApplied Biosytems TaqMan One-Step RT-PCR Master Mix Reagents kit. Suchreagents can be used to establish the expression ratio of ε, γ, and βusing purified RNA from enriched samples. Forward primers can be labeledfor each of the targets, using Alexa fluor-355 for ε, Alexa fluor-488for γ, and Alexa fluor-555 for β. Enriched cells can be deposited bycytospinning onto glass slides. Additionally, cytospinning the enrichedcells can be performed after in situ RT-PCR. Thereafter, the presence ofthe fluorescent-labeled amplicons can be visualized by fluorescencemicroscopy. The reverse transcription time and PCR cycles can beoptimized to maximize the amplicon signal:background ratio to havemaximal separation of fetal over maternal signature. Preferably,signal:background ratio is greater than 5, 10, 50 or 100 and the overallcell loss during the process is less than 50, 10 or 5%.

Fetal Cell Analysis

The detection and analysis steps may involve quantifying genomic DNAregions from cells in a sample or enriched sample. In some embodiments,the quantified genomic DNA regions are polymorphic sites such as shorttandem repeats (STRs) or variable number of tandem repeats (VNTRs).

In step 103, polymorphic genomic DNA region(s) or whole genome(s) fromthe mixed sample and optionally reference sample are pre-amplified toincrease the overall abundance of DNA used for quantification andanalysis. Pre-amplification can be preformed using multiple displacementamplification (MDA) (Gonzalez et al. Envircon Microbiol; 7(7); 1024-8(2005)) or amplification with outer primers in a nested PCR approach.This permits detection and analysis of fetal DNA even if the totalamount of fetal DNA in the mixed (e.g. enriched) sample is only up to 1μg, 500 ng, 200 ng, 100 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 1ng, 500 pg, 200 pg, 100 pg, 50 μg, 40 pg, 30 pg, 20 p, 10 pg, 5 pg, or 1pg or between 1-5 μg, 5-10 μg, or 10-50 μg. Pre-amplification allows theproducts to be split into multiple reactions at the next step.

In step 104, polymorphic DNA region(s) such as short tandem repeats(STRs) or variable number of tandem repeats (VNTRs) are selected onsuspected trisomic chromosome(s) (e.g., 13, 18, 21, X or Y) orchromosome(s) associated with a condition to be detected and optionallyon control (non-trisomic) chromosomes. In some embodiments, 1 or morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 DNA polymorphic loci areselected per target chromosome. Multiple polymorphic regions can beanalyzed independently or at the same time in the same reaction. Thepolymorphic DNA regions, e.g. STRs loci, are selected for highheterozygosity (variety of alleles) so that the paternal allele of thefetal cells is more likely to be distinct in length from the maternalalleles. This results in an improved power to detect the presence offetal cells in the mixed sample and potential fetal abnormalities insuch cells. When the polymorphic regions selected are STR loci, di-,tri-, tetra- or penta-nucleotide repeat loci can be used for detectionand analysis of fetal cells. Examples of STR loci that may be selectedinclude: D21S1414, D21S1411, D21S1412, D21S11 MBP, D13S634, D13S631,D18S535, AmgXY, XHPRT, as well as those listed in FIG. 12 . In someembodiment, the methods of the invention allow for the determination ofmaternal or paternal trisomy.

In step 105, the polymorphic loci selected are amplified. This can beused to detect non-maternal fetal alleles in the mixed sample and todetermine the copy number of such alleles. When amplifying more than onepolymorphic loci or DNA regions, primers are selected to bemultiplexable (fairly uniform melting temperature, absence ofcross-priming on the human genome, and absence of primer-primerinteraction based on sequence analysis) with other primer pairs. Primersand loci are chosen so that the amplicon lengths from a given locus donot overlap with those from another locus.

In some embodiments, multiple dyes and multi-color fluorescence readoutmay be used to increase the multiplexing capacity, e.g. of a single CGE.This ensures that the loci are kept distinct in the readout (e.g. CGEreadout). In such a case, PCR primer pairs can be grouped and the sameend-labeling is applied to the members of a group.

Examples of primers known in the art that correspond to specific STRloci that can be used in the present invention are described in FIG. 13and FIG. 14 .

Examples of PCR techniques that can be used to amplify the DNA regionsherein include, but are not limited, to quantitative PCR, quantitativefluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real timePCR (RT-PCR), single cell PCR, restriction fragment length polymorphismPCR (PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, in situpolonony PCR, in situ rolling circle amplification (RCA), bridge PCR,picotiter PCR and emulsion PCR. Other suitable amplification methodsinclude the ligase chain reaction (LCR), transcription amplification,self-sustained sequence replication, selective amplification of targetpolynucleotide sequences, consensus sequence primed polymerase chainreaction (CP-PCR), arbitrarily primed polymerase chain reaction(AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) and nucleicacid based sequence amplification (NABSA). Other amplification methodsthat may be used to amplify specific polymorphic loci include thosedescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and6,582,938.

In step 106, the amplified DNA polymorphic regions (e.g. STR loci) fromboth mixed and reference samples are characterized and quantified usingany method known in the art. Examples of such methods include, but arenot limited to, gas chromatography, supercritical fluid chromatography,liquid chromatography, including partition chromatography, adsorptionchromatography, ion exchange chromatography, size-exclusionchromatography, thin-layer chromatography, and affinity chromatography,electrophoresis, including capillary electrophoresis, capillary zoneelectrophoresis, capillary isoelectric focusing, capillaryelectrochromatography, micellar electrokinetic capillary chromatography,isotachophoresis, transient isotachophoresis and capillary gelelectrophoresis, comparative genomic hybridization (CGH), microarrays,bead arrays, high-throughput genotyping technology, such as molecularinversion probe (MIP), and Genescan.

In one embodiment, capillary gel electrophoresis (CGE) is used toquantify STRs in both the mixed and reference samples. This can be usedto detect non-maternal fetal alleles in the mixed sample and todetermine the copy number of such alleles. The mixed sample and thereference sample can be analyzed in separate reactions, e.g. separateCGE lanes. Alternatively, the mixed and the reference sample can be runin the same reaction, e.g. same CGE lane, by using two different dyelabels, e.g. differently labeled PCR primers. When a reference sample isrun through the PCR/CGE process, the alleles show up as peaks in theCGE. It is desirable, but not essential, to associate these peaks withknown alleles in the population at each locus. When performing PCR/CGEit may be very useful to reduce the non-linearities in the response ofPCR to input DNA copies (i.e. to effect more quantitative PCR) so thatthe data can be more easily related to models of aneuploidy. This‘linearization’ can be accomplished by the following procedure:

(a) The PCR reaction is initiated.

(b) The PCR reaction is interrupted after N cycles (N=5 to 10) and ˜onethird of the reaction products are removed and run on CGE. PCR cyclingis re-initiated. Repeat until 40 PCR cycles or saturation is achieved.

(c) CGE peak masses are determined and normalized to correct for thedepletion of the reaction products at each iteration of (b).

(d) A saturation (splining) curve is fit to the normalized data for eachallele peak, and quantitative starting concentrations are inferred as incustomary qPCR.

The above procedure tends to accomplish quantitative PCR while enablinga high degree of multiplexing. Because each CGE run has a slightlydifferent relation between DNA fragment size (and sequence) andmobility, each trace typically will need to undergo a lengthtransformation, such as a low-order (cubic or quartic) polynomialtransformation, in order to map to the data from the trace correspondingto the previous amplification point. This mapping can be determined byadjusting the transformation parameters to achieve the best fit of theone data trace to the other, with both normalized to the same total sumof squares or summed peak heights.

The maternal peaks at each locus provide an estimate of the secondary‘stutter’ structure at each locus due to PCR errors. The locations ofthese small secondary peaks can be used to blank out length regions thatare contaminated by this stutter when looking for and using thenon-maternal allele peaks (as described herein for example).Alternatively, more sophisticated ‘deconvolution’ algorithms can beapplied to remove the stutter Stoughton, et al., Electrophoresis; 18(1):1-S(1997).

The sample containing an unknown mixture of fetal and maternal cells isanalyzed as in Step (b). This could be done in a separate CGE lane, orin the same CGE lane as the maternal sample by using two different dyelabels on the PCR primers. Because each CGE trace has a slightlydifferent relation between DNA fragment size (and sequence) andmobility, these data typically will need to undergo a lengthtransformation, such as a low-order (cubic or quartic) polynomialtransformation in order to map one trace onto the other to facilitatepeak identification and model fitting. This mapping can be determined byadjusting the transformation parameters to achieve the best fit of thepeak locations in one data trace to the other. This mapping will be welldetermined in the assumed situation where the maternal cells are morenumerous than the fetal cells, because the maternal signature willdominate and will be shared in the two data sets.

FIG. 5 illustrates typical locus patterns arising from a normal(diploid) fetus and mother. At Locus 1, the paternal allele is the sameas the left hand maternal allele, and adds to its apparent height. AtLocus 2, the paternal allele has a length between the lengths of thematernal alleles. In addition, there is a secondary ‘stutter’ peak onthe shoulder of the right hand maternal peak. In Locus 3, the maternalsample is homozygous leading to only one main peak, and the paternalallele is distinct from this allele.

FIG. 6 illustrates locus patterns arising from trisomic fetal cells. Thedashed trace represents mixed sample containing trisomic fetal cells,superposed on maternal sample trace (solid black). Trisomy causes excessamplitude in maternal alleles at loci contained within the aneuploidregion (here assumed to contain Loci 1 and 2 but not Locus 3). The lefthand maternal peak at Locus 1 contains contributions from the trisomyand from a paternal allele.

In step 107, data models are constructed. From the data obtained fromthe quantifying step different data models can be constructed dependingupon different assumptions.

For example, a data model for the CGE patterns in FIGS. 5 and 6 can beas follows:

Let m1 denote the CGE signal obtained from one of the maternal allelesat a given locus and m2 the signal obtained from the other maternalallele, which might be the same allele. Let p denote the CGE signalobtained from the paternal allele at a given locus. Let p1 and p2 denotethe CGE signals obtained from the paternal alleles at a given locus whena paternally derived trisomy occurs. Let □ α and β denote the relativenumber of maternal and fetal cells, respectively. Then in the case of achromosome with maternal non-disjunction trisomy, the data will have theform

x=α(□m1+m2)+β(m1+m2+p).  (1)

A normal (diploid) chromosome will give

x=α(m1+m2)+β([m1 or m2]+p),  (2)

and a paternally derived trisomy will give

x=α(m1+m2)+β([m1 or m2]+p1+p2).  (3)

In some embodiments, data and data model is represented as discrete peakmasses (or heights) and peak locations or as vectors of valuesrepresenting the actual peak profiles. In the case of representation bypeak characteristics, the ‘addition’ operation in Equations 1-3 denotessummation of peak height or mass at the discrete allele location. In thecase where the full peak profiles are represented, summation denotessummation of signals bin by bin over the CGE trace, and in this case itmay be helpful to zero the data except in the immediate vicinity ofactual peaks. Representation via peak characteristics is preferable whenusing the PCR linearization technique described above.

To determine aneuploidy, the differences between the structure of theβ-term that appears in the first and second equations above isdetermined. In the first case, there is an additional contribution toboth maternal alleles along with the paternal allele, and in the secondcase there is an additional contribution only to one of the maternalalleles along with the paternal allele. The essence of thepresence/absence declaration for fetal cells lies in the evidence for βbeing greater than zero.

In step 108, the best overall fit of model to data is selected fromamong all the model sets. This modeling approach optimally usesinformation contained in the increase of chromosome copy number withaneuploidy and its association with the strength of non-maternalalleles.

In some embodiments, CGE signals representing m1 and m2 at each locusare obtained by profiling the maternal-only sample and mapping the peaklocations to the corresponding ones of the mixed sample. The heights ofm1 and m2 may be unequal, and this helps correct for PCR amplificationbiases associated with particular alleles. The values of p, p1, p2, α,and β are determined from the mixed sample data by fitting Equations 1-3to the data, optionally by using the least squares, or the maximumlikelihood methods.

The three models need to be fit to each chromosome with suspectedtrisomy, e.g. chromosomes 13, 18, 21, X and/or Y. If there are only 3suspected chromosomes, this results in 27 model variants (3×3×3=27). InEquations 2 and 3, there is also the ambiguity between using m1 or m2 inthe β-term, so there are 5 model variants for each chromosome, with5×5×5=125 total variants over three suspected trisomy chromosomes.

Segmental aneuploidies also could be tested by hypothesizing thatdifferent contiguous subsets of loci are contained within the aneuploidregion. With each model variant, α and β have to be determined and theparameters describing the paternal alleles have to be determined at eachlocus for each model variant. The paternal allele peak height and shapecan be assumed to be an average of the known maternal ones at thatlocus, while the paternal allele location needs to be fit to the data.The possible locations for the paternal allele will be the location ofm1, the location of m2, and ‘elsewhere in the locus window’ where thislatter possibility involves a search over discrete shifts smaller than atypical peak half-width at half maximum. Prior probabilities on thechoices of p, taken from population allele frequency data, can be used,if their product lengths can be predicted.

In some cases, because of the number of parameters being fit, suboptimalsearches can be used for computational efficiency. For example, onepossible approach involves iterative methods, such as the following,which would be applied to each data model variant:

(i) Set β to 0 and solve for α.

(ii) Set β to a value where β/α is the smallest fetal/maternal cellratio for which fetal cells are likely to be detectable.

(iii) Solve for paternal allele location(s) at each locus, one locus ata time that minimize data-model residuals.

(iv) Fix the paternal allele parameters and adjust β to minimizeresiduals over all the data.

(v) Now vary only a to minimize residuals.

(vi) Repeat iv and v until convergence.

(vii) Repeat iii through v until convergence.

In step 109, the presence or absence of fetal DNA is determined usingthe models described above. The best overall fit for such models yieldsthe values of β, α that can be called β_(max)□, α_(max). The likelihoodof observing the data given β_(max) can be compared to the likelihoodgiven β=0. The ratio is a measure of the amount of evidence for fetalDNA. A threshold for declaring fetal DNA is the likelihood ratio ofapproximately 1000 or more. The likelihood calculation can beapproximated by a Chi-squared calculation involving the sum of squaredresiduals between the data and the model, where each residual isnormalized by the expected rms error.

If it is determined that fetal DNA is not present in the mixed sample ascalculated above, then the test is declared to be non-informative. Onthe other hand, if it is decided that fetal DNA is present in the mixedsample, then the likelihoods of the data given the different data modeltypes can be compared to declare trisomy or another condition.

In step 110, the likelihood ratios of trisomy models (Equations 1 and 3)to the normal model (Equation 2) are calculated and these ratios arecompared to a predefined threshold. This threshold can be set so that incontrolled tests all the trisomic cases are declared aneuploid, and sothat it is expected that the vast majority (>99.9%) of all trulytrisomic cases are declared aneuploid by the test. In one embodiment, toaccomplish a detection rate of >90% or 95% or approximately 99.9%, thelikelihood ratio threshold is increased beyond what is necessary todeclare all the known trisomic cases in the validation set by a factorof 1000/N, where N is the number of trisomy cases in the validation set.

In step 111, errors that may arise from the experimental procedure usedto obtain the data can be taken into account in the modelcalculation(s). For instance, in the example described above, CGE datacontain small additive errors associated with CGE readout, and largermultiplicative errors associated with PCR amplification efficienciesbeing different from locus to locus and from allele to allele within alocus. By using the maternal-only data to define m1 and m2 peakcharacteristics at each locus, the effects of PCR amplification biasesassociated with different primers and different amplicons from the sameprimers have been mostly controlled. Nevertheless, small variations inthe process from day to day and the statistics of small numbers ofstarting genome copies will cause some random errors to remain. Thesetend to be multiplicative errors in the resulting CGE peak heights; e.g.two peaks may be 20% different in height although the startingconcentrations of the alleles were identical. In one embodiment, it maybe assumed that errors are random from peak to peak, and have relativelysmall additive errors, and larger Poisson and multiplicative errorcomponents. The magnitudes of these error components can be estimatedfrom repeated PCR/CGE processing of identical samples. The Chi-squareresiduals calculation for any data-model fit then can be supported withthese modeled squared errors for any peak height or data bin.

In another aspect of the present invention, the presence of fetal cellsin a mixed sample and fetal abnormalities in said cells is determinedwithout trying to integrate them in a data-model fitting proceduredescribed above. For example, steps 100-107 can be performed asdescribed above. Then, analysis using Equations 1 and 2 focuses on twoindications. First, aneuploidy results in an excess of DNA for thetrisomic chromosome, and this is indicated by the difference in meanstrengths of the alleles on the trisomic chromosome compared to controlchromosomes. A t-test can be applied to the two distributions of m1 andm2 peak heights. These peak heights are normalized to (e.g., dividedpeak-by-peak by) the corresponding peaks in the maternal-only sample toreduce PCR amplification biases. Second, Equations 1 and 2 show thataneuploidy is associated with less inequality in the heights of m1 andm2 at a given locus, particularly for loci where the paternal allele isdistinct from the maternal alleles. Loci are selected where a third(paternal) allele is visibly distinct from two maternal alleles, and thedistribution of the inequalities (measured in %) between the m1 and m2peaks are compared between suspected trisomy chromosomes and controlchromosomes. Again, peak heights first are normalized by thematernal-only sample. These two lines of evidence are combined to createan overall likelihood, such as by multiplying the probability valuesfrom the two lines of evidence. The presence/absence call is done in asimplified way by looking for loci where a third allele is clearlyvisible, and comparing the distribution of these peak heights betweenthe maternal and mixed samples. Again, a t-test between thesedistributions gives the probability of fetal DNA being present.

In another aspect of the invention, the methods herein only determinepresence or absence of fetal DNA, and aneuploidy information is knownfrom another sources (e.g. fluorescence in situ hybridization (FISH)assay). For example, it may be desirable only to verify the presence orabsence of fetal cells to ensure that a diploid test result is truly dueto a normal fetus and not to failure of an assay (e.g. FISH). In thiscase, the process may be simplified by focusing on detecting thepresence of non-maternal alleles without regard to associating them withincreases in the maternal allele strengths at the same locus. Thus aprocess similar to the one outlined above may be used but it is not asnecessary to arrange the PCR product lengths so that the products fromdifferent loci have distinct length windows in the CGE readout. Thealleles from the different loci can be allowed to fall essentiallyanywhere in the effective measurement length window of the CGE. It alsois not necessary to ‘lineate’ the PCR result(s) via multiple CGEreadouts at different stages in the PCR cycling as is suggested in step107.

Therefore, maternal-only and mixed samples are run and mapped to eachother to align maternal allele peak locations, as described above. PCRis run to saturation, or nearly to saturation, to be sure to detect thelow abundance fetal sequences. The evidence for fetal DNA then arisesfrom extra peaks in the mixed-sample data with respect to thematernal-sample data. Based on typical heterozygosities of approximately0.7 for highly polymorphic STRs, the chance of not seeing a distinctpaternal allele (distinct from both maternal alleles) when fetal DNA isin fact present decreases approximately as (0.7²)^(N), where N is thenumber of loci included. Thus approximately ten STR loci will provide˜99.9% probability of detection. In addition, the present inventionprovides methods to determine when there are insufficient fetal cellsfor a determination and report a non-informative case. The presentinvention involves quantifying regions of genomic DNA from a mixedsample. More particularly the invention involves quantifying DNApolymorphisms from the mixed sample. In some embodiments, one or morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 DNA polymorphism loci(particularly STRs) per target chromosome are analyzed to verifypresence of fetal cells.

Any of the steps above can be performed by a computer program productthat comprises a computer executable logic that is recorded on acomputer readable medium. For example, the computer program can executesome or all of the following functions: (i) controlling enrichment offetal cells or DNA from mixed sample and reference sample, (ii)pre-amplifying DNA from both samples, (iii) amplifying specificpolymorphic DNA regions from both samples, (iv) identifying andquantifying maternal alleles in the reference sample, (v) identifyingmaternal and non-maternal alleles in the mixed sample, (vi) fitting dataon alleles detected from mixed and/or reference samples into datamodels, (vii) determining the presence or absence of fetal cells in themixed sample, (viii) declaring normal or abnormal phenotype for a fetusbased on data models or declaring non-informative results, and (ix)declaring a specific fetal abnormality based on the above results. Inparticular, the computer executable logic can fit data on the quantityof DNA polymorphism(s) (e.g. STR's) into one or more data models. Oneexample of a data model provides a determination of a fetal abnormalityfrom given data signals obtained by molecular analysis e.g. CGE. Thecomputer executable logic provides for a determination of the presenceor absence of a trisomy, and distinguish whether the trisomy ispaternally derived or if it originates from a maternal non-disjunctionevent. For example, given the following data signals that can beobtained by molecular analysis (e.g. CGE)

m1, which represents a signal obtained from one of the maternal alleles(m1) at a given locus,

m2, which represents a signal obtained from the other maternal allele,which might be the same allele,

p, which is a signal that is obtained from the paternal allele at agiven locus, and

p1 and p2, which are signals obtained from the paternal alleles at onegiven locus when a paternally derived trisomy occurs, and

letting α and β, which denote the relative number of maternal and fetalcells, respectively, the following determinations can be made. In thecase of a chromosome with maternal non-disjunction trisomy, the datawill have the form

x=α(□m1+m2)+β(m1+m2+p).  (1)

A normal (diploid) chromosome will give

x=α(m1+m2)+β([m1 or m2]+p),  (2)

and a paternally derived trisomy will give

x=α(m1+m2)+β([m1 or m2]+p1+p2).  (3)

The computer executable logic can work in any computer that may be anyof a variety of types of general-purpose computers such as a personalcomputer, network server, workstation, or other computer platform now orlater developed. In some embodiments, a computer program product isdescribed comprising a computer usable medium having the computerexecutable logic (computer software program, including program code)stored therein. The computer executable logic can be executed by aprocessor, causing the processor to perform functions described herein.In other embodiments, some functions are implemented primarily inhardware using, for example, a hardware state machine. Implementation ofthe hardware state machine so as to perform the functions describedherein will be apparent to those skilled in the relevant arts.

The program can provide a method of evaluating the presence or absenceof trisomy in a mixed cell sample by accessing data that reflects thelevel of polymorphism(s) at two alleles at two or more given loci in amixed sample (maternal and fetal cells) and in a sample enriched infetal cells, relating the levels of polymorphism(s) to the number ofmaternal and fetal cells (α and β in equations 1-3), and determining thepresence or absence of trisomy in the samples.

In one embodiment, the computer executing the computer logic of theinvention may also include a digital input device such as a scanner. Thedigital input device can provide information on the polymorphismlevels/quantity. For example, a scanner of this invention can provide animage of the DNA polymorphism (particularly STRs) according to methodherein. For instance, a scanner can provide an image by detectingfluorescent, radioactive, or other emission; by detecting transmitted,reflected, or scattered radiation; by detecting electromagneticproperties or other characteristics; or by other techniques. The datadetected is typically stored in a memory device in the form of a datafile. In one embodiment, a scanner may identify one or more labeledtargets. For instance, a first DNA polymorphism may be labeled with afirst dye that fluoresces at a particular characteristic frequency, ornarrow band of frequencies, in response to an excitation source of aparticular frequency. A second DNA polymorphism may be labeled with asecond dye that fluoresces at a different characteristic frequency. Theexcitation sources for the second dye may, but need not, have adifferent excitation frequency than the source that excites the firstdye, e.g., the excitation sources could be the same, or different,lasers.

Another aspect of the invention includes kits containing the devices andreagents for performing the enrichment and genetic analysis. Such kitsmay include the materials for any individual step disclosed, anycombination of devices and reagents or the devices and reagents forperforming all of the steps. For example, a kit may include the arraysfor size-based enrichment, the device for magnetic separation of thecells and reagents for performing PCR or CGE. Also included may be thereagents for performing multiple displacement amplification. This is anexemplary kit and the kits can be constructed using any combination ofdisclosed materials and devices. The use of the size-based enrichmentprovides gentle handling that is particularly advantageous forpermitting subsequent genetic analysis.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1. Separation of Fetal Cord Blood

FIGS. 7A-7D illustrates a schematic of the device used to separatenucleated cells from fetal cord blood.

Dimensions: 100 mm×28 mm×1 mm

Array design: 3 stages, gap size=18, 12 and 8 μm for the first, secondand third stage, respectively.

Device fabrication: The arrays and channels were fabricated in siliconusing standard photolithography and deep silicon reactive etchingtechniques. The etch depth is 140 μm. Through holes for fluid access aremade using KOH wet etching. The silicon substrate was sealed on theetched face to form enclosed fluidic channels using a blood compatiblepressure sensitive adhesive (9795, 3M, St Paul, Minn.).

Device packaging: The device was mechanically mated to a plasticmanifold with external fluidic reservoirs to deliver blood and buffer tothe device and extract the generated fractions.

Device operation: An external pressure source was used to apply apressure of 2.0 PSI to the buffer and blood reservoirs to modulatefluidic delivery and extraction from the packaged device.

Experimental conditions: Human fetal cord blood was drawn into phosphatebuffered saline containing Acid Citrate Dextrose anticoagulants. 1 mL ofblood was processed at 3 mL/hr using the device described above at roomtemperature and within 48 hrs of draw. Nucleated cells from the bloodwere separated from enucleated cells (red blood cells and platelets),and plasma delivered into a buffer stream of calcium and magnesium-freeDulbecco's Phosphate Buffered Saline (14190-144, Invitrogen, Carlsbad,Calif.) containing 1% Bovine Serum Albumin (BSA) (A8412-100 ML,Sigma-Aldrich, St Louis, Mo.) and 2 mM EDTA (15575-020, Invitrogen,Carlsbad, Calif.).

Measurement techniques: Cell smears of the product and waste fractions(FIG. 8A-8B) were prepared and stained with modified Wright-Giemsa(WG16, Sigma Aldrich, St. Louis, Mo.).

Performance: Fetal nucleated red blood cells were observed in theproduct fraction (FIG. 8A) and absent from the waste fraction (FIG. 8B).

Example 2. Isolation of Fetal Cells from Maternal Blood

The device and process described in detail in Example 1 were used incombination with immunomagnetic affinity enrichment techniques todemonstrate the feasibility of isolating fetal cells from maternalblood.

Experimental conditions: blood from consenting maternal donors carryingmale fetuses was collected into K₂EDTA vacutainers (366643, BectonDickinson, Franklin Lakes, N.J.) immediately following electivetermination of pregnancy. The undiluted blood was processed using thedevice described in Example 1 at room temperature and within 9 hrs ofdraw. Nucleated cells from the blood were separated from enucleatedcells (red blood cells and platelets), and plasma delivered into abuffer stream of calcium and magnesium-free Dulbecco's PhosphateBuffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1%Bovine Serum Albumin (BSA) (A8412-100 ML, Sigma-Aldrich, St Louis, Mo.).Subsequently, the nucleated cell fraction was labeled with anti-CD71microbeads (130-046-201, Miltenyi Biotech Inc., Auburn, Calif.) andenriched using the MiniMACS™ MS column (130-042-201, Miltenyi BiotechInc., Auburn, Calif.) according to the manufacturer's specifications.Finally, the CD71-positive fraction was spotted onto glass slides.

Measurement techniques: Spotted slides were stained using fluorescencein situ hybridization (FISH) techniques according to the manufacturer'sspecifications using Vysis probes (Abbott Laboratories, Downer's Grove,Ill.). Samples were stained from the presence of X and Y chromosomes. Inone case, a sample prepared from a known Trisomy 21 pregnancy was alsostained for chromosome 21.

Performance: Isolation of fetal cells was confirmed by the reliablepresence of male cells in the CD71-positive population prepared from thenucleated cell fractions (FIG. 9 ). In the single abnormal case tested,the trisomy 21 pathology was also identified (FIG. 10 ).

Example 3. Confirmation of the Presence of Male Fetal Cells in EnrichedSamples

Confirmation of the presence of a male fetal cell in an enriched sampleis performed using qPCR with primers specific for DYZ, a marker repeatedin high copy number on the Y chromosome. After enrichment of fnRBC byany of the methods described herein, the resulting enriched fnRBC arebinned by dividing the sample into 100 PCR wells. Prior to binning,enriched samples may be screened by FISH to determine the presence ofany fnRBC containing an aneuploidy of interest. Because of the lownumber of fnRBC in maternal blood, only a portion of the wells willcontain a single fnRBC (the other wells are expected to be negative forfnRBC). The cells are fixed in 2% Paraformaldehyde and stored at 4° C.Cells in each bin are pelleted and resuspended in 5 μl PBS plus 1 μl 20mg/ml Proteinase K (Sigma #P-2308). Cells are lysed by incubation at 65°C. for 60 minutes followed by inactivation of the Proteinase K byincubation for 15 minutes at 95° C. For each reaction, primer sets (DYZforward primer TCGAGTGCATTCCATTCCG; DYZ reverse primerATGGAATGGCATCAAACGGAA; and DYZ Taqman Probe6FAM-TGGCTGTCCATTCCA-MGBNFQ), TaqMan Universal PCR master mix, NoAmpErase and water are added. The samples are run and analysis isperformed on an ABI 7300: 2 minutes at 50° C., 10 minutes 95° C.followed by 40 cycles of 95° C. (15 seconds) and 60° C. (1 minute).Following confirmation of the presence of male fetal cells, furtheranalysis of bins containing fnRBC is performed. Positive bins may bepooled prior to further analysis.

FIG. 20 shows the results expected from such an experiment. The data inFIG. 20 was collected by the following protocol. Nucleated red bloodcells were enriched from cord cell blood of a male fetus by sucrosegradient two Heme Extractions (HE). The cells were fixed in 2%paraformaldehyde and stored at 4° C. Approximately 10×1000 cells werepelleted and resuspended each in 5 μl PBS plus 1 μl 20 mg/ml ProteinaseK (Sigma #P-2308). Cells were lysed by incubation at 65° C. for 60minutes followed by a inactivation of the Proteinase K by 15 minute at95° C. Cells were combined and serially diluted 10-fold in PBS for 100,10 and 1 cell per 6 μl final concentration were obtained. Six μl of eachdilution was assayed in quadruplicate in 96 well format. For eachreaction, primer sets (DYZ forward primer TCGAGTGCATTCCATTCCG; 0.9 uMDYZ reverse primer ATGGAATGGCATCAAACGGAA; and 0.5 uM DYZ TaqMan Probe6FAM-TGGCTGTCCATTCCA-MGBNFQ), TaqMan Universal PCR master mix, NoAmpErase and water were added to a final volume aof 25 μl per reaction.Plates were run and analyzed on an ABI 7300: 2 minutes at 50° C., 10minutes 95° C. followed by 40 cycles of 95° C. (15 seconds) and 60° C.(1 minute). These results show that detection of a single fnRBC in a binis possible using this method.

Example 4. Confirmation of the Presence of Fetal Cells in EnrichedSamples by STR Analysis

Maternal blood is processed through a size-based separation module, withor without subsequent MHEM enhancement of fnRBCs. The enhanced sample isthen subjected to FISH analysis using probes specific to the aneuploidyof interest (e.g., triploidy 13, triploidy 18, and XYY). Individualpositive cells are isolated by “plucking” individual positive cells fromthe enhanced sample using standard micromanipulation techniques. Using anested PCR protocol, STR marker sets are amplified and analyzed toconfirm that the FISH-positive aneuploid cell(s) are of fetal origin.For this analysis, comparison to the maternal genotype is typical. Anexample of a potential resulting data set is shown in Table 2.Non-maternal alleles may be proven to be paternal alleles by paternalgenotyping or genotyping of known fetal tissue samples. As can be seen,the presence of paternal alleles in the resulting cells, demonstratesthat the cell is of fetal origin (cells #1, 2, 9, and 10). Positivecells may be pooled for further analysis to diagnose aneuploidy of thefetus, or may be further analyzed individually.

TABLE 2 STR locus alleles in maternal and fetal cells STR STR STR STRSTR locus locus locus locus locus DNA Source D14S D16S D8S F13B vWAMaternal alleles 14, 17 11, 12 12, 14 9, 9 16, 17 Cell #1 alleles 8 19Cell #2 alleles 17 15 Cell #3 alleles 14 Cell #4 alleles Cell #5 alleles17 12 9 Cell #6 alleles Cell #7 alleles 19 Cell #8 alleles Cell #9alleles 17 14 7, 9 17, 19 Cell #10 alleles 15

Example 5. Confirmation of the Presence of Fetal Cells in EnrichedSamples by SNP Analysis

Maternal blood is processed through a size-based separation module, withor without subsequent MHEM enhancement of fnRBCs. The enhanced sample isthen subjected to FISH analysis using probes specific to the aneuploidyof interest (e.g., triploidy 13, triploidy 18, and XYY). Samples testingpositive with FISH analysis are then binned into 96 microtiter wells,each well containing 15 μl of the enhanced sample. Of the 96 wells, 5-10are expected to contain a single fnRBC and each well should containapproximately 1000 nucleated maternal cells (both WBC and mnRBC). Cellsare pelleted and resuspended in 5 μl PBS plus 1 μl 20 mg/ml Proteinase K(Sigma #P-2308). Cells are lysed by incubation at 65° C. for 60 minutesfollowed by a inactivation of the Proteinase K by 15 minute at 95° C.

In this example, the maternal genotype (BB) and fetal genotype (AB) fora particular set of SNPs is known. The genotypes A and B encompass allthree SNPs and differ from each other at all three SNPs. The followingsequence from chromosome 7 contains these three SNPs (r57795605,rs7795611 and rs7795233 indicated in brackets, respectively)(ATGCAGCAAGGCACAGACTAA[G/A]CAAGGAGA[G/C]GCAAAATTTTC[A/G]TAGGGGAGAGAAATGGGTCATT).

In the first round of PCR, genomic DNA from binned enriched cells isamplified using primers specific to the outer portion of thefetal-specific allele A and which flank the interior SNP (forward primerATGCAGCAAGGCACAGACTACG; reverse primer AGAGGGGAGAGAAATGGGTCATT). In thesecond round of PCR, amplification using real time SYBR Green PCR isperformed with primers specific to the inner portion of allele A andwhich encompass the interior SNP (forward primerCAAGGCACAGACTAAGCAAGGAGAG; reverse primerGGCAAAATTTTCATAGGGGAGAGAAATGGGTCATT).

Expected results are shown in FIG. 31 . Here, six of the 96 wells testpositive for allele A, confirming the presence of cells of fetal origin,because the maternal genotype (BB) is known and cannot be positive forallele A. DNA from positive wells may be pooled for further analysis oranalyzed individually.

Example 6. Analysis of STR's

FIG. 11 illustrates a diagram of the planned protocol for clinicalpractice where a reference sample (maternal blood) and a fetal enrichedsample will be processed in parallel. Twelve polymorphic STRs are chosen(See FIG. 12 ) and associated nested PCR primers were designed (FIG.13-14 ).

Cell Lysis: Cells are lysed in a proteinase K solution by heatingsamples for 60 minutes at 65° C., followed by a heating step of 15minutes at 95° C.

1^(st) round of PCR: A polymerase mix that includes 12 specialized STRprimer pairs is added to the crude lysate. A master PCR mix is generatedaccording to the number of samples as per the recipe below. For thereference sample 44 μL of the master mix are added directly to the celllysate. For the sample recovered from the slide the volume of thereaction is adjusted as necessary. (e.g. 32 μL crude lysate in a 100 μLtotal reaction volume). A no template or negative control is generatedto test for contamination.

Master mix outer 12-plex Multiplex PCR cylce 1 rxn Step Temp (C.) Time(mins) 2X Qiagen Mix 25.0 1.0 95 0.5 titanium 1.0 2.0 94 0.5 Qiagen Qfactor 5.0 3.0 68 1.5 water 10.0 4.0 72 1.5 4.2 uM 12 plex primers 3.05.0 cycle to step 2, 44 times Cell lysate 6.0 6.0 72 10 50.0

Nested PCR: After PCR, optionally, diluted products are added to asecond nested primer PCR reaction. Two ul aliquot of each 12-plex PCRreaction is diluted 40 fold (to 80 ul total) with nuclease free waterfrom the PCR kit. The diluted fetal enriched 12-plex reaction could beused as template for a master mix for 8 nested PCR reactions with FAMlabeled primers. A second master mix can be generated using the dilutionfrom the maternal reference for 8 nested PCR reactions with VIC labeledprimers. The following primer pairs are suggested. A no template ornegative control is generated to test for contamination.

Nested STR primer facts Reaction Fragment size rxn# temp STR primersranges 1 68 CSF1P0 THO1 295-327, 171-215 2 68 TPOX CYARO4 220-256,172-205 3 68 F13A 179-235  4 68 FIBRA 158-286  5 63 VWA D21S11 122-182202-265 6 63 CD4 86-141 7 63 D14S1434 70-102 8 63 D22S1045 76-109

Master mix for nested primers 1 rxn 9 rxns 2X Q Mix 12.5 112.5 titanium0.5 4.5 Q 2.5 22.5 water 3.3 29.3 5 uM primers 1.3 40X diluted template5.0 45.0 25.0 213.8

Nested PCR cycle Step Temp (C.) Time (mins) 1.0 95 0.5 2.0 94 0.5 3.0 X1.5 4.0 72 1.5 5.0 cycle to step 2, 44 times 6.0 72 10

The amplification with the nested PCR primers is run with an annealingtemperature of 63° C. or 68° C. depending on the primer pair beingamplified as indicated in FIG. 13 and FIG. 14 .

Detection on ABI 310 Instrument: PCR products are detected on an AgilentBioanalyzer. The maternal reference VIC labeled PCR reaction products isdiluted 10 fold in nano-pure water (17.8 uOhms). Another 10 folddilution of the fetal enriched FAM-PCR products is generated. The ABIloading buffer is prepared by adding 0.5 μL LIZ 500 size standard to 12μL Hi Di Formamide (scale as appropriate to the number of samples,include enough buffer for the negative control to test forcontamination). 1 μL diluted PCR product is added to 12 ul loadingbuffer. The sample is heated to 95° C. for 2 minutes and then placed onice. The samples are loaded onto the ABI 310 as per the manufacturer'sinstructions.

Analysis: For analysis the ABI fragments output are examined for theexpected peak sizes as per the nested STR primer facts table (FIG. 13and FIG. 14 ). For each STR locus is determined whether there are 1 or 2alleles (homozygous or heterozygous) for the fetal enriched (FAM labeledsample) or the maternal reference (VIC labeled sample). Allelesgenerated from the fetal enriched (FAM labeled sample) that are notpresent in the maternal reference (VIC labeled sample) are unique to thefetus and verify the presence of fetal cells in the sample. If thenumber of fetal cells is particularly low (<5 cells), not all loci oralleles will always amplify. Allele drop out can generate a falsenegative. A false positive is most likely generated from contaminationand has not been observed in tests to date. If the purity of fetal cellsis particularly low (<10% in tests executed on the bioanalyzer) signalintensity of paternal alleles can be very weak. This can also generate afalse negative result. In clinical practice the amplicons will havedifferent fluorescent labels incorporated into them marking them as thematernal reference or the fetal enriched samples. The labels allow thesamples to be loaded simultaneously into an ABI 310 capillary anddifferentiated. Paternal alleles are identified as those unique to thefetal enriched sample in comparison to the maternal reference. Theefficiency of the overall process is determined after sufficient sampleshave been analyzed on the ABI 310 to establish the input cell purity andminimum number of fetal cells necessary to achieve 99% detection at 0.1%false positive rate.

Example 7. Cord Blood Experiment

The protocol detection limit was determined using fixed cells from cordblood. Cord blood from clinical sample SVH0003C was subjected toerythrocyte lysis and the remaining leukocytes were fixed in a solutionof PBS and 2% para-formaldehyde. Cell numbers were estimated fromhemocytometer counts and dilutions made into a Tris protienase K (PK)solution. After cell lysis and PK inactivation a PCR cocktail includingprimers for STR loci TPDX and CSF1P0 was added directly to the crudelysate and amplified as described in Example 6. The products wereanalyzed on an Agilent bioanalyzer. FIGS. 15A-15B shows representativeresults. TPDX and CSF1P0 amplification products are underline in boxes.Detection for the PCR protocol from fixed cord blood can occur with lessthan 10 cells as shown by the result in FIG. 16 .

Example 8. Detection of 10 Fetal Cells at 10% Purity without Nested PCR

An estimated 10 fetal cells were mixed with increasing amounts ofmaternal cells (approximately 0-4000 cells as measured by hemocytometercounts). After proteinase K lysis, only a 1^(st) round of PCR withprimers to STR loci D21S11 and VWA was executed as described in Example6. FIG. 17 shows representative results. The mother and fetus areidentical at the D21S11 locus but the child has a unique (paternal)allele at the VWA locus. FIG. 17 shows that detection of the paternalVWA allele is lost when the fetal purity drops below 10%.

Example 9. Generation of STR Markers

Approximately 100 cells were spotted onto a poly-L-lysine slide and heatdried. Cells were fixed in a MeOH acetic acid solution and rinsed inMeOH. After air drying the slide was treated with 2% para-formaldehydefor 10 minutes then washed in 1×PBS. The slide was dehydrated in passesof EtOH for 1 min each in 70%, 80%, 90%, and finally 100%. A dam wasapplied around the cells and 30 ul of proteinase K was added on top ofthe cells and a cover slip adhered over the dam. The slide was incubatedon a heat block at 65° C. for 60 minutes and 95° C. for 15 minutes. Thelysate solution was then transferred directly to a 100 ul PCR reactionwith VWA and LIPOL primer. PCR protocol and analysis were performed asdescribed in Example 6. FIG. 18 shows representative results. VWA andLIPOL amplicons are underlined by boxes in the figure. These resultsshow that STRs markers can be generated from fixed cells recovered froma slide.

Example 10. Detection of Fetal Alleles at Less than 10% Purity afterNested PCR Amplification of STRs

Mutliplex PCR reactions from samples with 10 fetal cells in a backgroundof maternal cells generating at 10%, 5% and 2% fetal cells purityconcentration were performed as described in Example 6. A dilution ofthe three multiplex PCR reactions was used as template in nested PCRreactions for STRs TPDX/CYAR04, VWA/D21S11, and D14S1434 as described inExample 6. FIG. 19 shows representative results. The underlined loci areknown to have unique fetal alleles which are designated by arrows whenvisible in the gel. The three loci were visible when fetal cellsconstituted 10% of the sample. D14S1434 loci was visible when fetalcells constituted 5% and 2% of the sample.

Example 11. Resolution of the Bioanalyzer

FIG. 15A shows that the Bioanalyzer can resolve a 16 base pairdifference between the 99 base pair D14S1434 maternal allele and the 83base pair paternal allele in a mixed fetal sample at 5% purity (alsoshown in FIG. 11 , lane 6). FIG. 15B shows that the output of fragmentscalibration standards. The output of an ABI310 fragment calibrationstandard is shown in blue or FAM label. Fragments of 232, 234, 236 and238 are easily resolved from one another. For a resolution comparisonnote that the orange, VIC labelled, 246 size standard peak is 8 basesaway from the FAM labeled 238 peak, half the distance as the maternaland paternal alleles in the Bioanalyzer trace.

Example 12. Analysis of STR's Using Quantitative Fluorescence

Genomic DNA from enriched fetal cells and a maternal control sample willbe genotyped for specific STR loci in order to assess the presence ofchromosomal abnormalities, such as trisomy. Due to the small number offetal cells typically isolated from maternal blood it is advantageous toperform a pre-amplification step prior to analysis, using a protocolsuch as improved primer extension pre-amplification (IPEP) PCR. Celllysis is carried out in 10 ul High Fidelity buffer (50 mM Tris-HCL, 22mM (NH.sub.4).sub.2 SO.sub.4 2.5 mM MgCl.sub.2, pH 8.9) which alsocontained 4 mg/ml proteinase K and 0.5 vol % Tween 20 (Merck) for 12hours at 48° C. The enzyme is then inactivated for 15 minutes at 94° C.Lysis is performed in parallel batches in 5 ul, 200 mM KOH, 50 mMdithiothreitol for 10 minutes at 65.degree. The batches are thenneutralized with 5 ul 900 mM TrisHCl pH 8.3, 300 mM KCl. Preamplicationis then carried out for each sample using completely randomized 15-merprimers (16 uM) and dNTP (100 uM) with 5 units of a mixture of Taqpolymerase (Boehringer Mannheim) and Pwo polymerase (BoehringerMannheim) in a ratio of 10:1 under standard PCR buffer conditions (50 mMTris-HCL, 22 mM (NH.₄)₂ SO₄, 2.5 mM Mg₂, pH 8.9, also containing 5% byvol. of DMSO) in a total volume of 60 ul with the following 50 thermalcycles: Step Temperature Time (1) 92° C. 1 Min 30 Sec; (2) 92° C. 40 Min(3) 37° C. 2 Min; (4) ramp: 0.1° C./sec to 55° C. (5) 55° C. 4 Min (6)68°) C. 30 Sec (7) go to step 2, 49 times (8) 8° C. 15′ Min.

Dye labeled primers will then be selected from Table 3 based on STR locion a chromosomes of interest, such as 13, 18, 21 or X. The primers aredesigned so that one primer of each pair contains a fluorescent dye,such as ROX, HEX, JOE, NED, FAM, TAMARA or LIZ. The primers are placedinto multiplex mixes based on expected product size, fluorescent tagcompatibility and melting temperature. This allows multiple STR loci tobe assayed at once and yet still conserves the amount of initialstarting material required. All primers are initially diluted to aworking dilution of 10 pM. The primers are then combined in a cocktailthat has a final volume of 40 ul. Final primer concentration isdetermined by reaction optimization. Additional PCR grade water is addedif the primer mix is below 40 ul. A reaction mix containing 6 ul ofSigma PCR grade water, 1.25 ul of Perkin Elmer Goldamp PCR buffer, 0.5ul of dNTPs, 8 ul of the primer cocktail, 0.12 ul of Perkin Elmer TaqGold Polymerase and 1.25 ul of Mg (25 mM) is mixed for each sample. Tothis a 1 ul sample containing preamplified DNA from enriched fetal cellsor maternal control genomic DNA is added.

The reaction mix is amplified in a DNA thermocyler, (PTC-200; MJResearch) using an amplification cycle optimized for the meltingtemperature of the primers and the amount of sample DNA.

The amplification product will then analyzed using an automated DNAsequencer system, such as the ABI 310, 377, 3100, 3130, 3700 or 3730, orthe Li-Cor 4000, 4100, 4200 or 4300. For example when the amplificationproducts are prepared for analysis on a ABI 377 sequencer, 6 ul ofproducts will be removed and combined with 1.6 ul of loading buffer mix.The master loading buffer mix contains 90 ul deionized formamidecombined with 25 ul Perkin Elmer loading dye and 10 ul of a sizestandard, such as the ROX 350 size standard. Various other standards canbe used interchangeably depending on the sizes of the labeled PCRproducts. The loading buffer and sample are then heat denatured at 95°C. for 3 minutes followed by flash cooling on ice. 2 ul of theproduct/buffer mix is then electrophoresed on a 12 inch 6% (19:1)polyacrylamide gel on an ABI 377 sequencer.

The results will then be analyzed using ABI Genotyper software. Theincorporation of a fluorochrome during amplification allows productquantification for each chromosome specific STR, with 2 fluorescentpeaks observed in a normal heterozygous individual with an approximateratio of 1:1. By comparison in trisomic samples, either 3 fluorescentpeaks with a ratio of 1:1:1 (trialleleic) or 2 peaks with a ratio ofaround 2:1(diallelic) are observed. Using this method screening may becarried out for common trisomies and sex chromosome aneuploidy in asingle reaction.

TABLE 3 Primer Sets for STRs on Chromsomes 13, 18, 21 and X STR Ch.Marker Primer 1 Primer 2 13 D13S317 5ACAGAAGTCTGGGATGTGGA (SEQ IDGCCCAAAAAGACAGACAGAA(SEQ NO 1) ID NO 2) D13S1493ACCTGTTGTATGGCAGCAGT (SEQ ID AGTTGACTCTTTCCCCAACTA (SEQ ID NO 3) NO 4)D13S1807 TTTGGTAAGAAAAACATCTCCC (SEQ GGCTGCAGTTAGCTGTCATT (SEQ IDID NO 5) NO 6) D13S256 CCTGGGCAACAAGAGCAAA (SEQ IDAGCAGAGAGACATAATTGTG (SEQ ID NO 7) NO 8) D13S258-ACCTGCCAAATTTTACCAGG (SEQ ID GACAGAGAGAGGGAATAAACC NO 9) (SEQ ID NO 10)D13S285 ATATATGCACATCCATCCATG (SEQ ID GGCCAAAGATAGATAGCAAGGTA NO 11)(SEQ ID NO 12) D13S303 ACATCGCTCCTTACCCCATC (SEQ IDTGTACCCATTAACCATCCCCA (SEQ NO 13) ID NO 14) D13S317ACAGAAGTCTGGGATGTGGA (SEQ ID GCCCAAAAAGACAGACAGAA(SEQ NO 15) ID NO 16)D13S779 AGAGTGAGATTCTGTCTCAATTAA GGCCCTGTGTAGAAGCTGTA (SEQ ID(SEQ ID NO 17) NO 18) D13S787 ATCAGGATTCCAGGAGGAAA (SEQ IDACCTGGGAGGCGGAGCTC (SEQ ID NO 19) NO 20) D13S793 GGCATAAAAATAGTACAGCAAGCATTTGAACAGAGGCATGTAC (SEQ ID (SEQ ID NO 21) NO 22) D13S796CATGGATGCAGAATTCACAG (SEQ ID TCATCTCCCTGTTTGGTAGC (SEQ ID NO 23) NO 24)D13S800 AGGGATCTTCAGAGAAACAGG (SEQ ID TGACACTATCAGCTCTCTGGC (SEQ NO 25)ID NO 26) D13S894 GGTGCTTGCTGTAAATATAATTG CACTACAGCAGATTGCACCA (SEQ ID(SEQ ID NO 27) NO 28) 18 D18S51 CAAACCCGACTACCAGCAAC (SEQ IDGAGCCATGTTCATGCCACTG (SEQ ID NO 29) NO 30) D18S1002CAAAGAGTGAATGCTGTACAAACAGC CAAGATGTGAGTGTGCTTTTCAGGAG (SEQ ID NO 31)(SEQ ID NO 32) D18S1357 ATCCCACAGGATGCCTATTT (SEQ IDACGGGAGCTTTTGAGAAGTT (SEQ ID NO 33) NO 34) D18S1364TCAAATTTTTAAGTCTCACCAGG GCCTGTAGAAAGCAACAACC (SEQ ID (SEQ ID NO 35)NO 36) D18S1370 GGTGACAGAGCAAGACCTTG (SEQ IDGCCTCTTGTCATCCCAAGTA (SEQ ID NO 37) NO 38) D18S1371CTCTCTTCATCCACCATTGG (SEQ ID GCTGTAAGAGACCTGTGTTG (SEQ ID NO 39) NO 40)D18S1376 TGGAACCACTTCATTCTTGG (SEQ ID ATTTCAGACCAAGATAGGC (SEQ ID NO 41)NO 42) D18S1390 CCTATTTAAGTTTCTGTAAGG (SEQ IDATGGTGTAGACCCTGTGGAA (SEQ ID NO 43) NO 44) D18S499CTGCACAACATAGTGAGACCTG (SEQ AGATTACCCAGAAATGAGATCAGC ID NO 45)(SEQ ID NO 46) D18S535 TCATGTGACAAAAGCCACAC (SEQ IDAGACAGAAATATAGATGAGAATGCA NO 47) (SEQ ID NO 48) D18S535TCATGTGACAAAAGCCACAC (SEQ ID AGACAGAAATATAGATGAGAATGCA NO 49)(SEQ ID NO 50) D18S542 TTTCCAGTGGAAACCAAACT (SEQ IDTCCAGCAACAACAAGAGACA (SEQ ID NO 51) NO 52) D18S843GTCCTCATCCTGTAAAACGGG (SEQ ID CCACTAACTAGTTTGTGACTTTGG NO 53)(SEQ ID NO 54) D18S851 CTGTCCTCTAGGCTCATTTAGC (SEQ IDTTATGAAGCAGTGATGCCAA (SEQ ID NO 55) NO 56) D18S858AGCTGGAGAGGGATAGCATT (SEQ ID TGCATTGCATGAAAGTAGGA (SEQ ID NO 57) NO 58)D18S877 GATGATAGAGATGGCACATGA (SEQ ID TCTTCATACATGCTTTATCATGC NO 59)(SEQ ID NO 60) 21 D21S11 GTGAGTCAATTCCCCAAG (SEQ ID NOGTTGTATTAGTCAATGTTCTCC 61) (SEQ ID NO 62) D21S1411ATGATGAATGCATAGATGGATG (SEQ AATGTGTGTCCTTCCAGGC (SEQ ID ID NO 63) NO 64)D21S1413 TTGCAGGGAAACCACAGTT (SEQ ID NO TCCTTGGAATAAATTCCCGG (SEQ ID 65)NO 66) D21S1432 CTTAGAGGGACAGAACTAATAGGC AGCCTATTGTGGGTTTGTGA (SEQ ID(SEQ ID NO 67) NO 68) D21S1437 ATGTACATGTGTCTGGGAAGG (SEQ IDTTCTCTACATATTTACTGCCAACA NO 69) (SEQ ID NO 70) D21S1440GAGTTTGAAAATAAAGTGTTCTGC CCCCACCCCTTTTAGTTTTA (SEQ ID (SEQ ID NO 71)NO 72) D21S1446 ATGTACGATACGTAATACTTGACAA GTCCCAAAGGACCTGCTC (SEQ ID NO(SEQ ID NO 73) 74) D21S2052 GCACCCCTTTATACTTGGGTG (SEQ IDTAGTACTCTACCATCCATCTATCCC NO 75) (SEQ ID NO 76) D21S2055AACAGAACCAATAGGCTATCTATC TACAGTAAATCACTTGGTAGGAGA (SEQ ID NO 77)(SEQ ID NO 78) X SBMA TCCGCGAAGTGAAGAAC (SEQ ID NOCTTGGGGAGAACCATCCTCA (SEQ ID 79) NO 80) DXS1047CCGGCTACAAGTGATGTCTA (SEQ ID CCTAGGTAACATAGTGAGACCTTG NO 81)(SEQ ID NO 82) DXS1068 CCTCTAAAGCATAGGGTCCA (SEQ IDCCCATCTGAGAACACGCTG (SEQ ID NO 83) NO 84) DXS1283EAGTTTAGGAGATTATCAAGCTGG GTTCCCATAATAGATGTATCCAG (SEQ ID NO 85)(SEQ ID NO 86) DXS6789 TTGGTACTTAATAAACCCTCTTTT CTAGAGGGACAGAACCAATAGG(SEQ ID NO 87) (SEQ ID NO 88) DXS6795 TGTCTGCTAATGAATGATTTGG (SEQ IDCCATCCCCTAAACCTCTCAT (SEQ ID NO 89) NO 90) DXS6800GTGGGACCTTGTGATTGTGT (SEQ ID CTGGCTGACACTTAGGGAAA (SEQ ID NO 91) NO 92)DXS6810 ACAGAAAACCTTTTGGGACC (SEQ ID CCCAGCCCTGAATATTATCA (SEQ ID NO 93)NO 94) DXS7127 TGCACTTAATATCTGGTGATGG (SEQ ATTTCTTTCCCTCTGCAACC (SEQ IDID NO 95) NO 96) DXS7132 AGCCCATTTTCATAATAAATCC (SEQ IDAATCAGTGCTTTCTGTACTATTGG NO 97) (SEQ ID NO 98) DXS8377CACTTCATGGCTTACCACAG (SEQ ID GACCTTTGGAAAGCTAGTGT (SEQ ID NO 99) NO 100)DXS9893 TGTCACGTTTACCCTGGAAC (SEQ ID TATTCTTCTATCCAACCAACAGC NO 101)(SEQ ID NO 102) DXS9895 TTGGGTGGGGACACAGAG (SEQ ID NOCCTGGCTCAAGGAATTACAA (SEQ ID 103) NO 104) DXS9896CCAGCCTGGCTGTTAGAGTA (SEQ ID ATATTCTTATATTCCATATGGCACA NO 105)(SEQ ID NO 106) DXS9902 TGGAGTCTCTGGGTGAAGAG (SEQ IDCAGGAGTATGGGATCACCAG NO 107) (SEQ ID NO 108) DXS998CAGCAATTTTTCAAAGGC (SEQ ID NO AGATCATTCATATAACCTCAAAAGA 109)(SEQ ID NO 110)

1-101. (canceled)
 102. A method of analyzing a fetal cell in a mixedsample obtained from a pregnant human female, the method comprising: (a)obtaining a mixed sample comprising fetal and maternal cells; (b)enriching the mixed sample for fetal cells to produce an enriched samplecomprising fetal cells and maternal cells, wherein the enrichmentincreases the ratio of fetal cells to maternal cells to about 1/10,000to about 1/10; (c) lysing fetal cells in the enriched sample andseparating nuclei of the fetal cells from other cells and cellcomponents in the enriched sample to produce isolated fetal nuclei; (d)isolating genomic DNA from the isolated fetal nuclei; (e) amplifyinggenomes of the isolated fetal nuclei to produce amplified nucleic acids;and (f) analyzing the amplified nucleic acids for aneuploidy usingultra-deep sequencing.
 103. The method of claim 102, wherein theanalyzing comprises analyzing for fetal aneuploidy, wherein the fetalaneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of oneor more chromosomes.
 104. The method of claim 103, wherein the fetalaneuploidy is a fetal aneuploidy of a chromosome selected from the groupconsisting of chromosome 13, chromosome 18, chromosome 21, chromosome X,and chromosome Y.
 105. The method of claim 103, wherein the fetalaneuploidy comprises trisomy or monosomy.
 106. The method of claim 105,wherein the fetal aneuploidy comprises trisomy, and wherein the trisomycomprises trisomy 13, trisomy 18, or trisomy
 21. 107. The method ofclaim 105, wherein the fetal aneuploidy comprises monosomy X and thechromosome suspected of being aneuploid comprises chromosome X.
 108. Themethod of claim 102, wherein the fetal aneuploidy comprises XXX, XXY, orXYY.
 109. The method of claim 102, wherein the ultra-deep sequencingproduces partial genome sequences for analysis.
 110. The method of claim102, wherein the ultra-deep sequencing produces complete genomesequences for analysis.
 111. The method of claim 102, wherein amplifyingthe genomes of the lysed fetal cells comprises amplifying whole genomesof the lysed fetal cells.
 112. The method of claim 102, furthercomprising binning fetal cells prior to the step of lysing fetal cellsin the enriched sample.
 113. The method of claim 112, wherein binningcomprises use of a nanofluidic system that separates samples intodroplets.
 114. The method of claim 102, further comprising positiveselection for fetal cells prior to binning.
 115. The method of claim102, further comprising negative selection for non-target cells prior tobinning.
 116. The method of claim 102, wherein the enrichment increasesthe ratio of fetal cells to maternal cells to about 1/100 to about 1/10.117. The method of claim 102, wherein the enrichment increases the ratioof fetal cells to maternal cells to about 1/50 to about 1/10.
 118. Themethod of claim 102, wherein the enrichment increases the ratio of fetalcells to maternal cells to about 1/10.
 119. The method of claim 102,wherein the mixed sample is obtained from whole blood, bone marrowsuspension, vaginal flow, milk, or genitourinary tracts fluid.
 120. Themethod of claim 102, wherein the enriching comprises contacting themixed sample with particles coupled to antibodies that selectively bindto fetal cells.
 121. The method of claim 120, wherein the particles aremagnetic particles.